This inention relates to enzyme-labeled antibody reagents which comprise an antibody reagent, such as whole native immunoglobulin or a fragment thereof, covalently linked to an enzyme. In particular, the invention concerns a method and coupling agent for preparing such labeled reagents in which the antibody reagent and enzyme portions retain substantially their native binding and catalytic properties, respectively.
Enzyme-labeled antibody reagents have a variety of uses, principally in the detection and measurement of antigens and haptens to which the antibody reagent portion is directed, and offer a safe and convenient alternative to the use of radioisotopically-labeled antibody reagents. An important analytical use of enzyme-labeled antibody reagents is the enzyme immunoassay method. Such method, as is well known in the art, can take a variety of forms or protocols. In general, a test sample to be assayed for the presence or amount of an antigenic or haptenic analyte is combined in one or more steps with reagents that include an enzyme-labeled component which ultimately is partitioned between bound and free-forms. The enzyme activity in either of the bound and free-forms can then be measured and related to the presence or amount o.f the analyte in the test sample.
Enzyme immunoassays which require the physical separation of the bound and free-forms of the labeled reagent are referred to as heterogeneous and are exemplified by the methods described in U.S. Pat. Nos. 3,654,090; 4,016,043; and Re. 31,006. Those which can be performed without physical separation of the bound and free-forms are referred to as homogeneous and are exemplified by the descriptions in U.S. Pat. Nos. 3,817,837 and 4,043,872. Particularly useful enzyme immunoassay protocols involving the use of labeled antibody reagents are those known commonly as the immunometric and sandwich techniques.
Aside from immunoassays, enzyme-labeled antibody reagents find use in any analytical method in which a substance having antigenic or haptenic properties is detected. Such substance can be the analyte of interest or related by some indirect or intermediary assay interaction to an analyte of interest. Examples are the detection and visualization of antigens in histological and cytological samples and the detection of antigenic and haptenic labels or antigenic hybrids in nucleic acid hybridization assays. The latter assays are exemplified by the methods described in published European Patent Specification Nos. 146,039 and 163,220 commonly assigned herewith.
All of the above methods and uses of enzyme-labeled antibody reagents are dependent on the ability to conveniently and reproducibly prepare the necessary conjugates of the desired enzyme and antibody reagent components. Furthermore, critical features of the labeled reagents are the binding and catalytic properties of the conjugated antibody and enzyme portions respectively. A variety of protein-protein coupling techniques are known in the literature and many have been applied to the preparation of enzyme-labeled antibody reagents. Recent review articles in this area include those by Peters and Richards, Ann. Rev. Biochem. 47:523(1977); Das and Cox, Ann. Rev. Biophys. Bioeng. 8:165(1979); Ji, Biochem. Biophys. Acta 559:39(1979); and Conn. Meth. in Enzymol. 103:49(1983). Typical homobifunctional linking reagents include amine-to-amine coupling agents, e.g., dimethyl imidates such as dimethyl adipimidate, dimethyl malonimidate, and dimethyl suberimidate; bis-N-oxysuccinimidyl esters such as disuccinimidyl suberate (DS) and disuccinimidyl tartarate; and bis-nitrofluorobenzenes such as 1,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'-dinitrophenylsulfone; sulfhydryl coupling agents, e.g., bis-maleimido reagents such as 1,2-phenylenedimaleimide and 1,4-phenylenedimaleimide; bis-iodoacetamides such as N,N-ethylene-bis-iodoacetamide; and bis-organomercury reagents such as 3,6-bis-(mercurimethyl)-dioxan; and the highly reactive diisothiocyanates such as 4,4'-diisothiocyano-2,2'-disulfonic acid and p-phenylene-diisothiocyanate (DTIC) and aryl azides such as 4,4'-dithio-bis-phenylazide.
Heterobifunctional coupling reagents are conceptually prepared by matching the above chemically compatible reactive groups. Some common examples are 4-fluoro-3-nitrophenylazide (FNPA), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ester.
Common disadvantages of bifunctional reagents are their sensitivity to moisture (e.g., adipimidate, N-oxysuccinimidyl (NOS) ester, and isothiocyanate reagents) or light (phenylazides). Some reagents are poorly soluble in water, a drawback which has been overcome in the case of N-hydroxysuccinimidyl ester reagents such as DS and MBS by the preparation of the N-3-sulfosuccinimidyl ester analogs. In addition, the spacer arms of most commonly used bifunctional reagents are either too short or too lipophilic, each affecting coupling efficiency and heterology. Further, conventional amine-amine coupling reagents have the disadvantage that the antibody component is generally quite susceptible to inactivation by reagents that react with primary amines.
The coupling of proteins through hydrophilic spacer groups is reviewed by Lowe and Dean, Affinity Chromatography, J. Wiley and sons (New York 1974), Chap. 5, pp. 200-259. Descriptions of particular hydrophilic spacer arms are provided by Porath, Meth. Enzymol. 34:24-27(1974) - bis-oxirane couplers; O'Carra et al, Meth. Enzymol. 34:116-118(1974) - 1,3-diaminopropan-2-ol; and Japanese Kokai Tokkyo Koho JP No. 58,176,547 (Chem. Abstr. 100:48087u) - polyethylene glycol diamines and dihydrazides. The use of aliphatic bis-maleimides as crosslinking agents is reviewed by Lundblad and Noyes, Chemical Reagents for Protein Modification, vol. 2, CRC Press (Boca Raton, Fla. 1984), Chap. 5, pp. 129-139; with specific reagents being exemplified by those described by Japanese Kokai Tokkyo Koho JP No. 58,183,094 (Chem. Abst. 100:99096d) and JP No. 58-49,821 (Chem. Abst. 100:135441y); Cooney et al, Biochem. Pharmocol. 27(2):151-166(1978); Heilmann and Holzner, BBRC 99:1146(1981); and Sato and Nakao, J. Biochem. 90:1177(1981).
Bis-maleimides have been used to couple enzymes, including .beta.-galactosidase, to antibody reagents [Yoshitake et al, Scand. J. Immunol. 10:81(1979)], but those that have been tried have been found to have such poor solubility in aqueous buffers that irreproducible syntheses result. There are no known attempts to use bis-maleimido polyalkyleneglycols as coupling agents for preparing enzyme-labeled antibody reagents, although such compounds are known, but have been used for completely unrelated purposes [see Japanese Kokai Tokkyo Koho JP No. 58-15,515 (Chem. Abst. 99:71625n), JP No. 58,136,637 (Chem. Abst. 100:104888v), and JP No. 58-40,374 (Chem. Abst. 99:124206k).